The objective of this project is to understand the mechanisms of ion permeation, ion selectivity, and gating in calcium release-activated calcium (CRAC) channels. CRAC channels play a vital role in the immune system by generating the sustained influx of calcium (Ca2+) into lymphocytes that is necessary for activation of immune-response genes. CRAC channels contain two major components: the protein Orai, which forms a Ca2+-conducting pore in the plasma membrane, and the regulatory protein STIM, which is a membrane protein located in the endoplasmic reticulum (ER). In a process known as gating, CRAC channels switch from a non- conductive (closed) state to a conductive (open) state in response to a reduction of the concentration of Ca2+ stored within the ER. Mutations in Orai that prevent gating cause severe combined immune deficiency, which underscores the importance of this process in immune function. Orai has also been implicated in cancer progression. In several respects, Orai is unlike other ion channels. My laboratory determined a 3-dimensional structure of Orai that represents a closed conformation of the channel. This `blueprint' of the channel revealed the architecture of Orai and its ion pore. With the proposed studies, we will investigate the channel's unprecedented mechanism of gating: the depletion of Ca2+ stored in the ER unites Orai and STIM across the cytosolic space separating their distinct membrane localizations, and this molecular `handshake' causes the ion pore in Orai to open. The opened channel is exquisitely selective for Ca2+ ions, effectively allowing only Ca2+ to enter the cell. The proposed studies also aim to address the mechanisms for how Ca2+ ions permeate through the channel and how the channel achieves such superb ion selectivity. This study uses X-ray crystallography to determine 3-dimensional structures of CRAC channels. Biochemical and biophysical techniques, including assays to measure CRAC channel function in cells and in reconstituted systems, will be used to correlate channel function with structural analysis. With these approaches we aim to: i) determine the 3-dimensional architecture of Orai in an open conformation, ii) visualize the molecular `handshake' between Orai and STIM that underlies gating, iii) investigate the mechanisms of highly selective Ca2+ permeation, and iv) investigate how small molecule inhibitors of CRAC channels inhibit the channel. The proposed studies will reveal principles of CRAC channel function, thereby making significant contributions to multiple fields of research including Ca2+ signaling, ion channels, and membrane protein structural biology. The studies will provide useful information for the design and optimization of CRAC channel inhibitors that could have utility as anti-cancer agents and as modulators of immune responses.